Process for Preparation of Cyclic Prodrugs of PMEA and PMPA

ABSTRACT

The method of preparing compounds of Formula I is described:  
                 
wherein: 
 
M and V are cis to one another and MPO 3 H 2  is a phosphonic acid selected from the group consisting of 9-(2-phosphonylmethoxyethyl)adenine, and (R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is phenyl, optionally substituted with 1-2 substituents selected from a group consisting of fluoro, chloro, and bromo; comprising: coupling a chiral 1-phenylpropane-1,3-diol, wherein the phenyl may be optionally substituted, with MPOCl 2  or an N-6 substituted analogue thereof. 
Additionally, methods and salt forms are described that enable isolation and purification of the desired isomer.

FIELD OF INVENTION

The present invention is directed towards a process of synthesis ofsubstituted six-membered cyclic 1-aryl-1,3-propanyl esters of PMEA andPMPA. More specifically, the invention relates to the process ofsynthesis of halogen substituted cyclic-1-phenyl-1,3-propanyl esters ofPMEA and PMPA that have cis stereochemistry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedto aid in understanding the invention, but is not admitted to be, or todescribe, prior art to the invention. All publications are incorporatedby reference in their entirety.

9-(2-phosphonylmethoxyethyl)adenine (PMEA),(R)-9-(2-phosphonyl-methoxypropyl)adenine (PMPA) and related analogues(U.S. Pat. No. 4,808,716; U.S. Pat. No. 5,142,051) are phosphonic acidsthat exhibit antiviral activity, including activity against hepatitis Band HIV (De Clercq et al., Antiviral Res. 8: 261-7(1987); Balzarini etal., Biochem Biophys. Res. Commun. 219(2): 337-41(1996)). Thedipivaloyloxy methylene ester of PMEA (“BisPOM PMEA”) is in clinicaltrials for the treatment of hepatitis B (Benhamou et al., Lancet358(9283): 718-23 (2001)). In addition, some studies have shown thatthese compounds also show anticancer activity (Murono et al., CancerRes. 61(21): 7875-7(2001)).

Compounds containing phosphonic acids and their salts are highly chargedat physiological pH and therefore frequently exhibit poor oralbioavailability, poor cell penetration and limited tissue distribution(e.g., CNS). In addition, these acids are also commonly associated withseveral other properties that hinder their use as drugs, including shortplasma half-life due to rapid renal clearance, as well as toxicities(e.g., renal, gastrointestinal, etc.) (e.g., Bijsterbosch et al.,Antimicrob Agents Chemother. 42(5): 1146-50 (1998)). Cyclic phosphonateesters have also been described for PMEA and related analogues. Thenumbering for these cyclic esters is shown below:

Unsubstituted cyclic 1′,3′-propanyl esters of PMEA were prepared butshowed no in vivo activity. EP 0 481214 B1 discloses examples of cyclicprodrugs of PMEA wherein the 1′ and 3′ positions are unsubstituted. Theapplication and a subsequent publication by the inventors (Starrett etal., J. Med. Chem. 37:1857-1864 (1994)) further disclose their findingswith the compounds, namely that these compounds showed no oralbioavailability and no biological activity. The compounds were shown tobe unstable at low pH, e.g., the cyclic 2′,2′-difluoro-1′,3′-propaneester is reported to be hydrolytically unstable with rapid generation ofthe ring-opened monoester.

SUMMARY OF THE INVENTION

The present invention is directed towards a novel process for thesynthesis of cyclic 1-aryl-1,3 propanyl phosphonate cyclic esters ofPMEA and PMPA with an enhanced d.e. for the cis isomer. In one aspectthe process enhances the cis isomers via a coupling method. In anotheraspect this process for the cis isomers is enhanced by the temperatureof the process. In an additional aspect the order of addition of thereactants enhanced the production of the cis isomer. Further aspect isadditional enrichment of the desired cis isomer through the addition ofan acid and the crystallization of the salt. Another aspect of theprocess is the enhancement of cis isomer that occurred with thecrystallization solvent.

In another aspect, this invention is directed towards a method of makingsubstantially enantiomerically pure cis cyclic esters having Sstereochemistry where the V is attached.

One aspect of the invention concerns the method for the preparation ofcompounds of Formula I:

wherein:

M and V are cis to one another and MPO₃H₂ is a phosphonic acid selectedfrom the group consisting of 9-(2-phosphonylmethoxyethyl)adenine, and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is phenyl,optionally substituted with 1-2 substituents selected from a groupconsisting of fluoro, chloro, and bromo; comprising: coupling a chiral1-phenylpropane-1,3-diol, wherein the phenyl may be optionallysubstituted, with MPOCl₂ or an N-6 substituted analogue thereof.

Additionally, methods and salt forms are described that enable isolationand purification of the desired isomer.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

The term “hexanes” refers to commercially available HPLC reagentsolutions which contains approximately 95% hexane, methylcyclopropane,and methylpentane.

The term “dialkyl” refers to a compound containing two alkyl groups. Theterm “alkyl” refers to saturated aliphatic groups includingstraight-chain, branched chain and cyclic groups. Suitable alkyl groupsinclude methyl, ethyl, isopropyl, and cyclopropyl.

The term “optionally substituted” or “substituted” includes aryl groupssubstituted with one to two substituents, independently selected fromlower alkyl lower aryl, and halogens. Preferably these substituents areselected from the group consisting of halogens.

The term “Cis” stereochemistry refers to the relationship of the V groupand M group positions on the six-membered ring. The formula below showsa cis stereochemistry.

Another cis stereochemistry would have V and M pointing above the plane.The formula below shows this cis stereochemistry.

The term “N6-substituted” refers to the substitution at the amineattached at the 6-position of a purine ring system. N6- is generallysubstituted with a dialkylaminomethylene group wherein R¹ groups includebut are not limited to C1-C4 acyclic alkyl, C5-C6 cyclic alkyl, benzyl,phenethyl, or R¹ groups together form piperdine, morpholine, andpyrrolidine.

The term “dialkylaminomethyleneimine” refers to functional group orsubstitution of the following structure:

wherein R¹ groups include but are not limited to C1-C4 acyclic alkyl,C5-C6 cyclic alkyl, benzyl, phenethyl, or R¹ groups together formpiperdine, morpholine, and pyrrolidine.

The term “percent enantiomeric excess (% ee)” refers to optical purity.It is obtained by using the following formula:${\frac{\lbrack R\rbrack - \lbrack S\rbrack}{\lbrack R\rbrack + \lbrack S\rbrack} \times 100} = {{\%\quad R} - {\%\quad S}}$

where [R] is the amount of the R isomer and [S] is the amount of the Sisomer. This formula provides the % ee when R is the dominant isomer.

The term “d.e.” refers to diastereomeric excess. It is obtained by usingthe following formula:${\frac{\lbrack{cis}\rbrack - \lbrack{trans}\rbrack}{\lbrack{cis}\rbrack + \lbrack{trans}\rbrack} \times 100} = {{\%\quad\lbrack{cis}\rbrack} - {\%\quad\lbrack{trans}\rbrack}}$

The term “diastereoisomer” refers to compounds with two or moreasymmetric centers having the same substituent groups and undergoing thesame types of chemical reactions wherein the diastereoisomers havedifferent physical properties, have substituent groups which occupydifferent relative positions in space, and may have different biologicalproperties.

The term “racemic” refers to a compound or mixture that is composed ofequal amounts of dextrorotatory and levorotatory forms of the samecompound and is not optically active.

The term “enantiomer” refers to either of a pair of chemical compoundswhose molecular structures have a mirror-image relationship to eachother.

The term “acid dissociation constant” (K_(a)) refers to the equilibriumconstant for the ionization of an acid, e.g. HA is the formula for aweak acid, then:K_(a)=([H⁺ ][A ⁻ ]/[HA])

The term “halogen” refers to chlorine, bromine, or fluorine.

The term “prodrug” as used herein refers to any M compound that whenadministered to a biological system generates a biologically activecompound as a result of spontaneous chemical reaction(s), enzymecatalyzed chemical reaction(s), and/or metabolic chemical reaction(s),or a combination of each. Standard prodrugs are formed using groupsattached to functionality, e.g., HO—, HS—, HOOC—, R₂N—, associated withthe drug, that cleave in vivo. Standard prodrugs include but are notlimited to carboxylate esters where the group is alkyl, aryl, aralkyl,acyloxyalkyl, alkoxycarbonyloxyalkyl as well as esters of hydroxyl,thiol and amines where the group attached is an acyl group, analkoxycarbonyl, aminocarbonyl, phosphate or sulfate. The groupsillustrated are exemplary, not exhaustive, and one skilled in the artcould prepare other known varieties of prodrugs. Such prodrugs of thecompounds of Formula I fall within the scope of the present invention.Prodrugs must undergo some form of a chemical transformation to producethe compound that is biologically active or is a precursor of thebiologically active compound. In some cases, the prodrug is biologicallyactive, usually less than the drug itself, and serves to improve drugefficacy or safety through improved oral bioavailability,pharmacodynamic half-life, etc. The biologically active compoundsinclude, for example, anticancer agents, and antiviral agents.

The term “cyclic 1′,3′-propane ester”, “cyclic 1,3-propane ester”,“cyclic 1′,3′-propanyl ester”, and “cyclic 1,3-propanyl ester” refers tothe following:

The term “enhancing” refers to increasing or improving a specificproperty.

The term “enriching” refers to increasing the quantity of a specificisomer produced by a reaction.

The term “pharmaceutically acceptable salt” includes salts of compoundsof Formula I derived from the combination of a compound of thisinvention and an organic or inorganic acid or base, such that they areacceptable to be safely administered to animals. Suitable acids includeacetic acid, adipic acid, benzenesulfonic acid,(+)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonic acid,citric acid, 1,2-ethanedisulfonic acid, dodecyl sulfonic acid, fumaricacid, glucoheptonic acid, gluconic acid, glucuronic acid, hippuric acid,hydrochloride hemiethanolic acid, HBr, HCl, HI, 2-hydroxyethanesulfonicacid, lactic acid, lactobionic acid, maleic acid, methanesulfonic acid,methylbromide acid, methyl sulfuric acid, 2-naphthalenesulfonic acid,nitric acid, oleic acid,4,4′-methylenebis[3-hydroxy-2-naphthalenecarboxylic acid], phosphoricacid, polygalacturonic acid, stearic acid, succinic acid, sulfuric acid,sulfosalicylic acid, tannic acid, tartaric acid, terephthalic acid, andp-toluenesulfonic acid.

The following well known chemicals are referred to in the specificationand the claims. Abbreviations, and common names are also provided.

-   -   CH₂Cl₂; Dichloromethane or methylene chloride    -   DCM; dichloromethane    -   (−)-DIP—Cl; (−)-β-Chlorodiisopinocampheylborane    -   DMAP; 4-dimethylaminopyridine    -   DMF; Dimethylformamide    -   HCl; hydrochloric acid    -   KI; potassium iodide    -   MgSO₄; magnesium sulfate    -   MTBE; t-butyl methyl ether    -   NaCl; sodium chloride    -   NaOH; sodium hydroxide    -   PyBOP; benzotriazol-1-yloxytripyrrolidinophosphonium        hexafluorophosphate    -   TEA; triethylamine    -   THF; tetrahydrofuran    -   TMSCl; chlorotrimethylsilane    -   bis POM PMEA;        bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl) adenine        (Adefovir dipivoxil)

The following well known drugs are referred to in the specification andthe claims. Abbreviations and common names are also provided.

-   -   PMEA; 9-(2-phosphonylmethoxyethyl)adenine (Adefovir)    -   (R)—PMPA; (R)-9-(2-phosphonylmethoxypropyl)adenine (Tenofovir)

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to the discovery that the process for thesynthesis of cyclic 1-aryl-1,3-propanyl esters of PMEA and PMPAdetermined the stereochemistry of the resultant product. Compoundssynthesized by the process of the present invention are directed towardsthe cis stereochemistry of the cyclic esters of PMEA and PMPA. In oneaspect of this invention the stereochemistry at the methine carbon whichis identified as C1′ in the cyclic 1-aryl-1,3-propanyl esters wasestablished through the synthesis of the corresponding chiral1-aryl-1,3-propane diol e.g., via the chiral reduction of anintermediate ketoacid.

In another aspect it was found that the chirality at the phosphorus ofthe cyclic phosphonate ring was established during the reaction with thediol. Production of the cis diastereoisomer was dependent on thereaction temperature and the order of addition of the chiral diol andprotected parent phosphonic dichloridate to the reaction mixture.

An additional aspect of the invention is the protection of the nitrogenattached to the carbon labeled 6 in the structure below.

The concentration of the desired cis isomer, wherein cis refers to thegeometric relationship between the phosphorus-carbon bond and thecarbon-aryl bond of the cyclic phosphonate ring, was enhanced byadditional isolation via selective crystallization of the acid salt,which is a further aspect of this invention. Still further enhancementwas achieved through recrystallization of the acid addition salt.

The process for the synthesis of cyclic 1-aryl-1,3-propanyl esters ofPMEA or PMPA with the desired stereochemistry is via a convergentsynthetic sequence starting with adenine and a halogen substitutedbenzoyl chloride. The final resultant compound contained twostereocenters, (1) the methine carbon which is identified as C1′ in thestereoisomeric structures and (2) the phosphorus of the cyclicphosphonate ring. The stereochemistry at the carbon, C1′, resulted fromthe chiral reduction of an intermediate ketoacid and the phosphoruschirality was the result of the diastereoselective coupling of theparent phosphonic acid and the chiral diol. The desired cis isomer,wherein cis refers to the isomeric relationship between thephosphorus-carbon bond and the carbon-phenyl bond of the cyclicphosphonate ring, was isolated via a selective crystallization of theacid salt.

Compounds Prepared by the Invention

The compounds prepared by the invention are substituted 6-memberedcyclic 1,3-propane diester prodrugs of PMEA and analogues as representedby Formula I:

wherein:

M and V are cis to one another and MPO₃H₂ is a phosphonic acid selectedfrom the group consisting of 9-(2-phosphonylmethoxyethyl)adenine, and(R)-9-(2-phosphonylmethoxypropyl) adenine;

V is phenyl, optionally substituted with 1-2 substituents selected froma group consisting of F, Cl, and Br;

and pharmaceutically acceptable salts thereof.

Another aspect of the invention is the preparation of the compounds ofFormula II

wherein:

MPO₃H₂ is a phosphonic acid selected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine and(R)-9-(2-phosphonylmethoxypropyl)adenine;

V is phenyl, optionally substituted with 1-2 substituents selected froma group consisting of F, Cl, and Br;

and pharmaceutically acceptable salts thereof.

Another aspect is directed to salts of such compounds formed withmethanesulfonic acid or succinic acid.

Another aspect is directed to salts formed with methanesulfonic acid.

Another aspect of the invention is the preparation of compounds ofFormula II

wherein:

MPO₃H₂ is a phosphonic acid selected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine and(R)-9-(2-phosphonylmethoxypropyl)adenine; V is 3-chlorophenyl;

and pharmaceutically acceptable salts thereof.

Another aspect is directed to salts formed with methanesulfonic acid ofsuch compounds.

Another aspect of the invention are the compounds of Formula II

wherein:

MPO₃H₂ is a phosphonic acid selected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine and(R)-9-(2-phosphonylmethoxypropyl)adenine; V is 2-bromophenyl;

and pharmaceutically acceptable salts thereof.

Another aspect is directed to salts formed with methanesulfonic acid ofsuch compounds.

1. Synthesis of 1-(Aryl)-Propane-1,3-Diols

A variety of synthetic methods are known to prepare 1,3-diols. Thesesuitable methods are divided into two types as following: 1) synthesisof racemic 1-(aryl)-propane-1,3-diol; 2) synthesis of chiral1-(aryl)-propane-1,3-diol.

1.1 Synthesis of Racemic 1-(Aryl)-Propane-1,3-Diol

1,3-Dihydroxy compounds can be synthesized by several well known methodsin literature. Substituted aromatic aldehydes are utilized to synthesizeracemic 1-(aryl)propane-1,3-diol via addition of lithium enolate ofalkyl acetate followed by ester reduction (path A) (Turner, J. Org.Chem. 55:4744 (1990)). Alternatively, aryl Grignard additions to1-hydroxy propan-3-al also give 1-(arylsubstitued)propane-1,3-diols(path B). This method will enable conversion of various substituted arylhalides to 1-(arylsubstituted)-1,3-propane diols (Coppi, et al., J. Org.Chem. 53:911 (1988)). Aryl halides can also be used to synthesize1-substituted propane diols by Heck coupling of 1,3-diox-4-ene followedby reduction and hydrolysis (Sakamoto, et al., Tetrahedron Lett. 33:6845(1992)). Pyridyl, quinoline, isoquinoline propan-3-ol derivatives can beoxygenated to 1-substituted-1,3-diols by N-oxide formation followed byrearrangement in acetic anhydride conditions (path C) (Yamamoto, et al.,Tetrahedron 37:1871 (1981)). A variety of aromatic aldehydes can also beconverted to 1-substituted-1,3-diols by vinyl Grignard addition followedby hydroboration reaction (path D).

1.2 Synthesis of Chiral 1-(aryl)-Propane-1,3-Diol

A variety of known methods for chiral resolution of secondary alcoholsvia chemical or enzymatic agents may be utilized for preparation of diolenantiomers (Harada, et al., Tetrahedron Lett. 28:4843 (1987)).Transition metal catalyzed hydrogenation of substituted 3-aryl-3-oxopropionic acids or esters is an efficient method to prepare R or Sisomers of optically pure beta hydroxy acids or esters (ComprehensiveAsymmetric Catalysis, Jacobsen, E. N., Pfaltz, A., Yamamoto, H. (Eds),Springer, (1999); Asymmetric Catalysis in Organic Synthesis, Noyori, R.,John Wiley, (1994)). These beta hydroxy acid or ester products can befurther reduced to give required chiral 1-(aryl)-propane-1,3-diols.(path A). The β-keto acid or ester substrates for high pressurehydrogenation or hydrogen transfer reactions may be prepared by avariety of methods such as condensation of acetophenone withdimethylcarbonate in the presence of a base (Chu, et al, J. Het Chem.22:1033 (1985)), by ester condensation (Turner, et al., J. Org. Chem.54:4229 (1989)) or from aryl halides (Kobayashi, et al., TetrahedronLett. 27:4745 (1986)). Alternatively, enantiomerically pure 1,3-diolscan be obtained by chiral borane reduction of β-hydroxyethyl aryl ketonederivatives or β-keto acid derivatives (path B) (Ramachandran, et al.,Tetrahedron Lett. 38:761 (1997)). In another method, commerciallyavailable cinnamyl alcohols may be converted to epoxy alcohols undercatalytic asymmetric epoxidation conditions. These epoxy alcohols arereduced by Red-Al to result in enantiomerically pure 1,3-diols (path C)(Gao, et al., J. Org. Chem. 53:4081 (1980)). Aldol condensation isanother well described method for synthesis of the chiral 1,3-oxygenatedfunctionality starting from aromatic aldehydes. (path D) (Mukaiyama,Org. React. 28:203 (1982)).

For the purpose of this invention the intermediate ketoacid is preparedfrom a halogen substituted benzoyl chloride of Formula A wherein thebenzoyl chloride may be optionally substituted at any position on thephenyl ring with 1-2 halogens. In a preferred embodiment if R² is ahalogen then R³ must be a hydrogen and if R³ is a halogen then R² mustbe a hydrogen. In one embodiment Formula A is 3-chlorobenzoyl chlorideand in another embodiment, Formula A is 2-bromobenzoyl chloride. The C1′identifies the carbon that is the methine carbon stereocenter in thefinal compound prepared by this invention.

The compound of Formula A is reacted with trimethylsilyl acetate andlithium diisopropylamide (generated in situ by reaction ofdiisopropylamine and n-butyllithium) to obtain the oxo-propanoic acid.The hydroxypropanoic acid is synthesized from the oxo-propanoic acid viareaction with (−)-DIP—Cl and then the hydroxypropanoic acid is reducedto the chiral 1,3-diol, shown in the following Formula B:

The chiral center at the carbon, C1′, has been established in thisprocess step and the ratio of enantiomers was conserved throughout theremainder of the process.

2.0 Synthesis of PMEA

Various preparations of PMEA and (R)-PMPA and their analogues aredescribed in the literature (Arimilli et al., WO 99/04774; Schultze etal., Tetrahedron Letters 1998, 39, 1853-1856; Bischofberger et al., U.S.Pat. No. 5,514,798, U.S. Pat. No. 5,686,629; Holy et al., U.S. Pat. No.4,659,825, U.S. Pat. No. 4,808,716, U.S. Pat. No. 5,130,427, U.S. Pat.No. 5,142,051) and are known to those skilled in the art. Theseprocedures were modified for use herein and the modifications wereunexpectedly found to eliminate both the time consuming isolation andpurification steps given in the earlier literature. For the purpose ofthis invention the isolation of the diethyl ester of the phosphonic acidwas not required to proceed to the next step. It was found that theester could be deprotected without purification in this process.

In a typical method the adenine is reacted with a substituted ornonsubstituted ethylene carbonate and a base to generate9-hydroxyethyladenine which was further alkylated with TsOCH₂P(O)OEt₂.The final step entailed a hydrolysis of the diethyl ester to generatePMEA, (R)-PMPA or their analogues.

3. Synthesis of N6-protected PMEA-dichloridate

In another step chlorination of PMEA is achieved using oxalyl chlorideand N,N-diethylformamide to give N6 protected-PMEA-dichloridate.N,N-dialkylformamide used in the chlorination step not only forms aVilsmeyer chlorinating agent, but also protects the NH₂ group at the 6position. The protected chloridate intermediate was found to havefavorable solubility properties that improved the overall yield and thediastereomeric ratio of the product. Use of other protecting groups suchas acyl, alkoxycarbonyl, aryloxycarbonyl, and aralkyloxycarbonyl alsoenhance the solubility of the dichloridate and diastereomeric ratio ofthe expected product.

4. Coupling of Phosphonic Dichloridate and Chiral Diol

Coupling of the protected parent phosphonic dichloridate and the chiraldiol in the presence of a base resulted in a protected intermediatesoluble in dichloromethane at lower temperatures.

4.1 Crystallization of cis Prodrug Salt

Deprotection of the N6 position of the coupled phosphonic acid andchiral diol under mild acidic conditions and crystallization of theresultant product using methanesulfonic acid gave rise to the cisprodrug as a mesylate salt (Formula C) with 92-93% chemical purity. Thetrans isomer is the major impurity and a second crystallization of thefinal material from an alcohol such as methanol gave greater than 96%diastereomeric purity.

The use of other acids including but not limited to such as sulfuric,nitric, hydrochloric, phosphoric, sulfonic, tartaric, citric, maleic,malic, malonic, lactic, oxalic acids and the like, may lead to betterrecovery and isomeric ratio of the product. The protocol as describedfor PMEA is also applicable to other PME or PMP derivatives.

4.2 Synthesis of9-{2-[2,4-cis-(S)-(+)-4-(halophenyl)-2-oxo-1,3,2-dioxa-phosphorinan-2-yl]methoxyethyl}adeninemesylate

The9-{2-[2,4-cis-(S)-(+)-4-(halophenyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]methoxyethyl}adeninemesylate (Formula C) was prepared via an eight step convergent syntheticsequence starting with adenine and halobenzoyl chloride. The finalresultant compound (Formula C) contained two stereocenters: (1) themethine carbon (C1′); and (2) the phosphorus of the cyclic phosphonatering. The stereochemistry at the carbon (C1′) resulted from the chiralreduction of the intermediate ketoacid and the phosphorus chirality wasthe result of the diastereoselective coupling of the parent phosphonicacid and the chiral diol. The desired cis isomer, wherein cis refers tothe isomeric relationship between the phosphorus-carbon bond and thecarbon-phenyl bond of the cyclic phosphonate ring, was isolated via aselective crystallization of the methanesulfonic acid salt.

The starting materials of the chiral diol and the parent phosphonic acidwere synthesized using modified procedures. The chiral diol wassynthesized from 3-chlorobenzoyl chloride via a three step sequence andthe parent phosphonic acid was synthesized from adenine via a four stepsequence.

The final desired (cis) stereoisomer product is obtained with highpurity via a novel coupling step wherein the parent phosphonic acid andthe chiral diol were coupled to produce the final stereoisomer product.

Previous coupling efforts wherein PMEA was reacted with racemic diolusing dehydrating agents such as, N,N′-dicyclohexylcarbodiimide andPyBOP in DMF/pyridine solvent systems, were found to require elevatedtemperatures (at least 100° C.) to achieve complete coupling. Thesereactions proceeded with a relatively minor diastereomeric excess (5-10%of the desired cis isomer). Unexpectedly and surprisingly, improvedd.e.'s were noted when the reaction temperature was lowered. This aspectof the invention led to an effort to activate PMEA as the dichloridate,a more reactive chemical species. It was the inventors' desire to reactthe dichloridate of PMEA with the diol at lower temperatures. Thedichloridate of PMEA is readily prepared using standard chlorinationconditions. The coupling reaction with the dichloridate at lowtemperature was complicated by the poor solubility of the dichloridate.Accordingly, the inventors sought protecting groups of N6 that would aidin the solubilization of the dichloridate. One preferred protectinggroup was the N-(dialkylaminomethyleneimine).

Formation of the N-(dialkylaminomethyleneimine)-protected PMEAdichloridate was achieved by treatment of PMEA in the presence of adialkylformamide, such as, dimethylformamide, diethylformamide,dibutylformamide, N-formylpiperidine, or N-formylmorpholine, with oxalylchloride in refluxing dichloromethane.

5.0 Effect of Dichloridate Addition Order and Temperature

The higher order formamides (diethyl and higher) gave a more lipophilicnature to the dichloridate. This lipophilic nature was found to make thedichloridate more soluble in dichloromethane (DCM). The addition of theracemic diol to the dichloridate intermediate, in the presence of anexcess of triethylamine (TEA), gave complete reaction but the reactionwas found to have only a modest d.e. In a preferred embodiment, when thereagents were added in reverse order (i.e. the dichloridate was added tothe diol/base mixture), an improved d.e. was obtained (cis:trans=71:29).Surprisingly the inventors found that the order of the addition and alow temperature produced a method for enriching the d.e. in favor of thecis isomer. The results are given in Table 1 (see entries 1-3).

TABLE 1 EFFECT OF TEMPERATURE AND ADDITION ORDER Temp Entry R GroupSolvent (° C.) Addition cis:trans 1 Methyl DCM −70 Dichloridate to diol75:25 2 Methyl DCM −50 Dichloridate to diol 71:29 3 Methyl DCM 0Dichloridate to diol 63:36 4 Methyl DCM −50 Diol to dichloridate 57:42 5Ethyl DCM −50 Base to mixture 66:34

Table 1 also shows that superior cis-trans ratios are achieved bylowering the temperature of the coupling reaction. See entries (1-3).

With the unexpected advantage of dichloridate being added to the diol,it was preferable for the dichloridate to remain in solution fortransferring. When R of the protecting group was methyl, the resultingdichloridate remained a slurry. Surprisingly, it was found that with anaddition of a slight excess of pyridine (1.1 equivalents), thedichloridate slurry dissolved. This may be due to the neutralization ofone equivalent of HCl and the resulting greater solubility of the freebase dichloridate versus the dichloridate hydrochloride.

The resulting crude reaction mixture was subjected to awater/dichloromethane partition work-up and the isolated couplingmixture was treated with refluxing acetic acid in ethanol to effectnitrogen deprotection.

5.1 cis Isomer Salt Formation and Solvents

When the coupling/deprotection sequence was performed with the chiraldiol (S or R), the same d.e. was observed (50%), and it was discoveredthat the cis enantiomer did not crystallize from the reaction solutionas did the cis racemate. Surprisingly, formation of certain salts of the75:25 cis:trans mixture led to a crystallization of the desired cisdiastereomer. A list of some salts that were used and the d.e.'s thatwere found of the solid and filtrates are listed in Table 2. TABLE 2 cisISOMER SALT AND CRYSTALLIZATION SOLVENTS SOLID FILTRATE SALT SOLVENT(cis:trans) (cis:trans) Free base 75:25 Succinic acid Ethanol 82:1870:30 L-Tartaric acid Ethanol 70:30 85:15 D-Tartaric acid Ethanol 76:2477:23 Maleic acid Ethanol 66:31 88:12 Methanesulfonic acidEthanol:acetone 93:7  57:43 L-Malic acid Ethanol:acetone 56:43 93:6 D-Malic acid Ethanol:acetone 56:43 95:2 

It was found that the methanesulfonic acid salt of the 75:25 cis:transmixture gave the highest enrichment of the desired cis diastereomer(93:7). Deprotection was conducted by refluxing with a weak acid, suchas acetic acid in an alcoholic solvent, e.g., ethanol. Methanesulfonicacid was then added to the reaction solution after deprotection wascomplete. At this stage it was found that the methanesulfonic acidselectively crystallized the desired cis diastereomer. The crudemesylate salt typically contained only 5-7% of the trans isomer, and afinal recrystallization was developed to further decrease the translevels to 1-3%. Table 3 lists some of the recrystallization solventsystems tried.

Using a sample containing 4% trans isomer dissolved in the solventslisted below, the cis isomer was enriched. TABLE 3 FINALRECRYSTALLIZATION SOLVENTS Solvent *Volume (mL)/g % Recovery A % TransMethanol 2.5 71.8 1.0 Ethanol 10.5 86.4 1.6 Isopropanol 48 88.4 2.2Methanol/Toluene (1/1) 5.0 58.8 1.0 Methanol/Isopropanol (1/1) 4.0 85.02.0 Methanol/Isopropanol (2/5) 7.0 89.2 2.5 Methanol/Ethanol (2/5) 7.083.4 2.7 Methanol/Acetone (1/1) 4.0 73.2 1.2*Volume used is per gram of sample.

The compounds used in this invention and their preparation can beunderstood further by the examples which illustrate some of theprocesses by which these compounds are prepared. These examples shouldnot however be construed as specifically limiting the invention andvariations of the compounds, now known or later developed, areconsidered to fall within the scope of the present invention ashereinafter claimed.

EXAMPLES Example 1 Preparation of 3-(3-Chlorophenyl)-3-oxo-propanoicacid (1)

A 12 L, 3-neck round bottom flask was equipped with a mechanical stirrerand addition funnel (2 L). The flask was flushed with nitrogen andcharged with diisopropylamine (636 mL) and THF (1.80 L). The stirredcontents were cooled to −20° C. n-Butyllithium (1.81 L of a 2.5 Msolution in hexanes) was added slowly with stirring, and the temperaturewas maintained between −20 and −28° C. After the addition was complete(30 min), the addition funnel was rinsed with hexanes (30 mL) and thestirred solution was then cooled to −62° C. Trimethylsilyl acetate (300g) was added slowly with stirring, maintaining the temperature at <−60°C. After the addition was complete (about 30 minutes), the solution wasstirred at −60° C. for 15 minutes. 3-Chlorobenzoyl chloride (295 mL) wasadded slowly with stirring, maintaining the temperature at <−60° C.After the addition was complete (about 65 minutes), the cooling bath wasremoved and the reaction solution was stirred for approximately 1.25hours, with gradual warming to 0° C. The reaction flask was cooled withan ice bath, then water (1.8 L) was added to the stirred solution. Thereaction mixture was stirred for 10 minutes, and then diluted witht-butyl methyl ether (MTBE) (1.0 L). The lower aqueous phase wasseparated and transferred to a round bottom flask equipped with amechanical stirrer. MTBE was added (1.8 L) and the stirred mixture wascooled to <10° C. in an ice bath. Concentrated HCl solution (300 mL of12 M solution) was added and the mixture was vigorously stirred. Thelayers were separated and aqueous phase was further acidified withconcentrated HCl (30 mL) and extracted again with MTBE (1.0 L). Thecombined MTBE extracts were washed with approximately 10% NaCl solution(1 L), dried (MgSO₄, 70 g), filtered and concentrated under reducedpressure to give 827 g of a yellow solid. The crude solid was slurriedin hexanes (2.2 L) and transferred to a round bottom flask equipped witha mechanical stirrer. The mixture was stirred at <10° C. for 1 hour,then filtered, washed with hexanes (4×100 mL) and dried to constantweight (−30 in. Hg, ambient temperature, 14 hours). The ¹H-NMR analysisfor this example and all following examples were performed on a VARIANGEMNI-200 MHz Spectrometer. The samples were dissolved in the indicatedsolvent and the chemical shifts are referenced to the residual solvent.

Recovery=309 g

Pale yellow powder 1 (68.6%).

¹H-NMR (acetone-d₆): δ=4.1 (s, 2H), 7.5-8.1 (m, 4H)

Example 2 Preparation of (S)-3-(3-Chlorophenyl)-3-hydroxypropanoic acid(2)

A 12 L, 3-neck round bottom flask was equipped with a mechanical stirrerand addition funnel (1 L). The flask was flushed with nitrogen andcharged with 3-(3-chlorophenyl)-3-oxo-propanoic acid (275.5 g) 1 anddichloromethane (2.2 L). A thermocouple probe was immersed in thereaction slurry and the stirred contents were cooled to −20° C.Triethylamine (211 mL) was added over 5 minutes to the stirred slurryand all solids dissolved. A dichloromethane solution of(−)-B-chlorodiisopinocampheylborane (1.60 M, 1.04 L) was charged to theaddition funnel, and then added slowly with stirring while maintainingthe temperature between −20 and −25° C. After the addition was complete(approximately 35 min), the solution was warmed to ice bath temperature(2-3° C.) and stirred. After approximately 4 hours of stirring anin-process NMR analysis indicated the starting material 1 was <4%.

The residual starting material 1 was measured by proton NMR as follows:removing a 0.5 mL sample of the reaction mixture and quenching withwater (0.5 mL) and 3 M NaOH solution (0.5 mL). The quenched mixture wasstirred and the layers separated. The aqueous phase was acidified with 2M HCl (1 mL) and extracted with ethyl acetate (1 mL). The organic phasewas separated, filtered through a plug of MgSO₄ and concentrated with astream of nitrogen. The residue was dissolved in CH₂Cl₂ and the solventwas evaporated with a stream of nitrogen. This residue was dissolved inacetone-d₆ and an analysis was done by ¹H-NMR(acetone-d₆).

Water (1.2 L) was added to the cloudy orange reaction mixture, followedby 3 M NaOH solution (1.44 L). The mixture was vigorously stirred for 5minutes and then transferred to a separatory funnel. The layers wereseparated and the basic aqueous phase was washed with ethyl acetate (1L). The aqueous phase was acidified with concentrated HCl (300 mL) andextracted with ethyl acetate (2 times with 1.3 L each). The two acidicethyl acetate extracts were combined, washed with approximately 10% NaClsolution (600 mL), dried with MgSO₄ (130 g), filtered and concentratedunder reduced pressure to provide 328 g of a yellow oil. The oilcrystallized upon standing. The resulting solid was slurried in ethylacetate (180 mL) and transferred to a 2 L, 3-neck round bottom flask,equipped with a mechanical stirrer. The stirred ethyl acetate mixturewas cooled to <10° C. (ice bath), then diluted with hexanes (800 mL).The resulting mixture was stirred at ice bath temperature for 4 h, andthen filtered. The collected solid was washed with 4:1 hexanes:ethylacetate (3×50 mL) and dried to constant weight (−30 inches of Hg,ambient temperature, 12 h).

Recovery=207.5 g

White powder 2 (74.5%)

¹H-NMR(acetone-d₆): δ=2.7 (d, J=6 Hz, 2H), 4.7 (d, J=4 Hz, 1H), 5.1-5.2(m, 1H), 7.2-7.5 (m, 4H).

Example 3 Preparation of (S)-(−)-1-(3-Chlorophenyl)-1,3-propanediol (3)

A 12 L, 3-neck round bottom flask was equipped with a mechanicalstirrer, addition funnel (2 L) and thermometer. The flask was flushedwith nitrogen and charged with (S)-3-(3-chlorophenyl)-3-hydroxypropanoicacid 2 (206.7 g) and THF (850 mL), and the stirred solution was cooledto 5° C. (ice bath). A 1 M borane in THF solution (2.14 L) was chargedto the addition funnel, and then added slowly with stirring maintainingthe temperature at <10° C. After the addition was complete(approximately 1 hour), the cooling bath was removed and the solutionwas stirred at ambient temperature for 1 hour. The reaction solution wasslowly and cautiously quenched with water (600 mL), followed by 3 M NaOHsolution (850 mL). The mixture was stirred for 10 minutes with anobserved temperature increase to approximately 40° C., and then themixture was transferred to a separatory funnel. The layers wereseparated and the aqueous phase was extracted again with ethyl acetate(600 mL). The combined organic phase was washed with approximately 10%NaCl solution (500 mL), dried (MgSO₄, 322 g), filtered and concentratedunder reduced pressure to provide 189.0 g of a pale yellow oil (101%).Preliminary analysis of the oil was by ¹H-NMR (CDCl₃).

The oil was purified by vacuum distillation and the fraction at 125-155°C./0.15 mmHg was collected.

Recovery=180.9 g

Colorless oil 3 (94.0%).

¹H-NMR (CDCl₃): δ=2.9-3.1 (m, 2H), 2.5 (bs, 2H), 3.9 (t, J=5 Hz, 2H),4.9(dd, J=7.4, 4.8 Hz, 1H), 7.2-7.4 (m, 4H).

Procedure for ee Determination

For the chiral HPLC analysis the diol 3 was derivatized to the diacetateas follows:

The resultant diol 3 (5.0 mg, 0.026 mmol) was dissolved indichloromethane (2.0 mL). Acetic anhydride (15 μL, 0.15 mmol) and4-(dimethylamino)pyridine (13 mg, 0.10 mmol) were added and the solutionwas stirred at ambient temperature for 15 minutes. The reaction solutionwas quenched with 1 M HCl solution (3 mL) and the lower organic phasewas separated, passed through a plug of MgSO₄, and concentrated with astream of nitrogen. The residue was dissolved in methanol (1 mL) andanalyzed by chiral HPLC. Surprisingly, the ee for the diol 3 wasdetermined to be >98%.

HPLC conditions:

Column: Pirkle covalent (S,S) Whelk-O 10/100 krom FEC, 250×4.6 mm;mobile phase=70:30, methanol:water, isocratic; flow rate=1.5 mL/min;injection volume=10 μL UV detection at 220 nm.

Retention times: S-diol (diacetate)=12.1 min, R-diol (diacetate)=8.6min.

Example 4 Preparation of Diethyl p-toluenesulfonyloxymethylphosphonate(4)

A 12 L, 3-neck round bottom flask was equipped with a mechanicalstirrer, condenser, thermometer and heating mantle. The flask wasflushed with nitrogen and charged with diethyl phosphite (554 g),paraformaldehyde (142 g), toluene (2 L) and triethylamine (53 mL). Themixture was stirred at 85-90° C. for 2 hours, and then refluxed for 1hour. The resulting yellow solution was cooled to 4° C. in an ice bathand p-toluenesulfonyl chloride (718 g) was added. The condenser wasreplaced with an addition funnel and triethylamine (750 mL) was addedslowly with stirring, maintaining the temperature at <10° C. After theaddition was complete (45 minutes), the resulting mixture was stirred atambient temperature for 14 hours. The mixture was filtered and thefiltercake was washed with toluene (2×250 mL). The combined filtrate andwashings were washed with water (2×1 L), dried (MgSO₄, 200 g), filteredthrough diatomaceous earth (Celite 521, CAS 61790-53-2), andconcentrated under reduced pressure.

Recovery=1004 g.

Cloudy yellow oil 4 (77.6%).

¹H-NMR (CDCl₃). Δ=1.3 (t, J=8H, m, 3H), 2.4(s, 3H), 4.0-4.2 (m, 4H), 7.2(d, J=8 Hz, 2H), 7.8 (d, J=8 Hz, 2H).

Example 5 Preparation of 9-(2-Hydroxyethyl)adenine (5)

A 12 L, 3-necked round bottom flask was equipped with a mechanicalstirrer, condenser, thermometer and heating mantle. The flask wasflushed with nitrogen and charged with adenine (504 g), ethylenecarbonate (343 g), DMF (3.7 L) and sodium hydroxide (7.80 g). Thestirred mixture was heated to reflux (approximately 80 minutes to reachreflux, pot temperature=145° C.), and then refluxed for 2 hours. Theheating mantle was removed and the yellow solution was cooled to below100° C. The resulting mixture was then cooled to 5° C. in an ice bathand diluted with toluene (3.8 L). The resulting mixture was stirred at<10° C. for 2 hours and then filtered. The collected solid was washedwith toluene (2×0.5) and cold ethanol (1.5 L), then dried to constantweight (−30 in. Hg, 50° C., 14 h).

The solid 5 was analyzed by HPLC and ¹H-NMR (DMSO-d₆).

HPLC conditions:

Silica column (particle size,10 microns) (Phenomenex Bondclone) 10 C18column, 300×3.9 mm; Mobile phase: Solvent A=20 mM potassium phosphate,pH 6.2; Solvent B=acetonitrile; Gradient: 0-60% B/15 min.,60-0% B/2min., 0% B/3 min.; UV detection at 270 nm.

Retention times: Product=6.5 min., 3-regioisomer (tentative)=5.6 min.

Recovery=624 g.

Pale yellow solid 5 (93.3%).

¹H-NMR (DMSO-d₆): δ=3.6-3.8 (m, 2H), 4.1 (t, J=6 Hz, 2H), 5.0 (bs, 1H),7.2(bs, 2H), 8.05(s, 1H), 8.10(s, 1H).

Example 6 Preparation of 9-(2-Diethylphosphonylmethoxyethyl)adenine (6)

A 5 L, 3-neck round bottom flask was equipped with a mechanical stirrerand thermometer. The flask was flushed with nitrogen and charged with9-(2-hydroxethyl)adenine 5 (464 g) and DMF (1.40 L). The stirred slurrywas cooled to 10° C. in an ice bath and sodium tert-butoxide (436 g) wasadded in one portion with a corresponding increase in temperature to 29°C. The ice bath was removed and the mixture was stirred at ambienttemperature for 1 hour yielding a slightly cloudy solution. The reactionflask was equipped with an addition funnel (2 L) and the stirredcontents were cooled to 5° C. (ice bath). Diethylp-toluenesulfonyloxymethylphosphonate (1130 g), as a solution in DMF(700 mL), was added slowly with stirring, maintaining the temperature at<10° C. After the addition was complete (2 hours), the cooling bath wasremoved and the mixture was stirred at ambient temperature for 1 hour.HPLC was used to determine completeness of the reaction. The mixture wassampled by removing 0.05 mL of the reaction mixture and dissolving thematerial in 10 mL of 20 mM potassium phosphate buffer, pH 6.2.

HPLC conditions:

Silica column (particle size, 10 microns) (Phenomenex Bondclone) 10 C18column, 300×3.9 mm; Mobile phase: Solvent A=20 mM potassium phosphate(pH 6.2, Solvent B=acetonitrile; Gradient: 0-60% B/15 min., 60-0% B/2min., 0% B/3 min.; UV detection at 270 nm; Injection volume=10 uL.

Retention times: Product 6=9.2 minutes, Starting material 5=6.5 minutes.

The stirred mixture was cooled to 10° C. and 80% acetic acid (250 mL)was slowly added. After the addition was complete (approximately 15minutes), the mixture was stirred at ambient temperature for 30 minutesand the temperature gradually increased to 30° C. The solvent wasevaporated under reduced pressure (R-152 rotary evaporator, 5 mm Hg) toprovide 2115 g of an orange sludge. The material was used withoutpurification for the next step.

Example 7 Preparation of 9-(2-Phosphonylmethoxyetnyl)adenine (7)

A 12 L, 3-neck round bottom flask was equipped with a mechanicalstirrer. The flask was charged with the crude9-(2-diethylphosphonylmethyoxyethyl)adenine 6, as a slurry inacetonitrile (4.0 L). The mixture was stirred at ambient temperature for30 minutes and then filtered. The filter cake was washed withacetonitrile (2×0.5 L) and the combined filtrate and washings were useddirectly as follows.

A 22 L, 3-neck round bottom flask was equipped with a mechanicalstirrer, thermometer, condenser and heating mantle. The flask wasflushed with nitrogen and charged with the9-(2-diethylphosphonylmethoxyethyl)adenine 6 solution (2.59 mol),chlorotrimethylsilane (1.315 L) and potassium iodide (1.719 kg). Therewas a gradual increase in temperature after the addition of KI to 35° C.The stirred mixture was then heated to 55° C. and stirred at 50-55° C.for 1 hour. The mixture was stirred for an additional 3 hours withgradual cooling to 38° C. HPLC was used to determine completeness of thereaction.

HPLC conditions:

Silica column (particle size,10 microns) (Phenomenex Bondclone) 10 C18,300×3.9 mm column; mobile phase: Solvent A=20 mM potassium phosphate, pH6.2, Solvent B=acetonitrile; Gradient: 0-60% B/15 min., 60-0% B/2 min.,0% B/3 min.; UV detection at 270 nm.

Retention times: Product 7=5.2 min., starting material 6=9.2 min.

The reaction flask was equipped with an addition funnel (2 L) and 3.5 MNaOH solution (4 L) was slowly added with a temperature increase from 32to 44° C. The two liquid phase system was transferred to a 5 gal.stationary separatory funnel and the layers were separated. The basicaqueous phase was extracted with ethyl acetate (2 L) and thentransferred to a 12 L, 3-neck flask, equipped with a mechanical stirrerand an addition funnel (1 L). Concentrated HCl was added slowly withstirring until the pH was 3.0 as determined by standard laboratory pHmeter. The resulting yellow solution was stirred at ambient temperaturefor 12 hours. A precipitate formed. The stirred mixture was cooled to 7°C. in an ice bath and the pH was readjusted to 3.0 with concentratedHCl. The mixture was stirred at ice bath temperature for 5 hours andthen filtered. Filtration took approximately 4 hours. The collectedsolid was washed with acetone and air dried on the filter funnel.

A 5 L, 3-neck round bottom flask was equipped with a mechanical stirrerand a 250 mL addition funnel. The flask was charged with the crude solidand 1 M sodium hydroxide solution (1.25 L). The mixture was stirreduntil all solids were dissolved (15 minutes). Concentrated HCl solutionwas added slowly to the stirred solution until the pH was 3.0. Theresulting mixture was stirred at ambient temperature for 4 hours andthen filtered. The collected solid was washed with water (2×250 mL) andacetone (200 mL), and dried to constant weight (−30 in. Hg, 60° C., 14hours).

Recovery=292 g

Off-white solid (41.3%).

¹H-NMR (D₂O); δ=3.25 (d, J=8 Hz, 2H), 3.70 (t, J=4 Hz, 2H), 4.10 (t, J=4Hz, 2H), 4.60 (s, 4H), 7.80 (s, 1H), 7.90 (s, 1H).

Example 8 Preparation 9-{2-[2,4-cis(S)-(+)-4-(3-Chlorophenyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]methoxyethyl}adeninemethanesulfonate (9) Example 8.1 Formation of Dichloridate (8)

A 2 L, 3-neck round bottom flask was equipped with a mechanical stirrer,condenser, addition funnel (125 mL) and heating mantle. The flask wasflushed with nitrogen and charged with PMEA 7 (50.0 g), dichloromethane(650 mL) and N,N-diethylformamide (22.5 mL). Oxalyl chloride (58.0 mL)was charged to the addition funnel, and added slowly to the stirredreaction mixture. Vigorous gas evolution occurred and the nitrogen inletwas removed to facilitate the gas to escape. After the addition wascomplete (15 minutes), the addition funnel was removed and thevigorously stirred mixture was heated at reflux for 2 hours. Thesolution remained a slurry during this process. The reaction mixture wascooled slightly, and additional oxalyl chloride (1.0 mL) andN,N-diethylformamide (0.4 ml) were added. The addition ofN,N-diethylformamide produced vigorous gas evolution. The resultingmixture was heated at reflux until all solids were dissolved (additional2.5 hours, total reaction time was approximately 4.5 hours). HPLCanalysis of the reaction solution indicated the product 8 at 83 Area %.The reaction was monitored for formation of the dichloridate. A sampleof the reaction mixture (approximately 50 μL) was removed and quenchedin anhydrous methanol (1 mL) containing 1 drop of triethylamine. Theresulting methyl phosphonate(s) were analyzed by HPLC.

HPLC conditions:

YMC-Pack R & D, R-33-5 S-5 120A, 250×4.6 mm; mobile phase: Solvent A=20mM potassium phosphate, pH 6.2; Solvent B=acetonitrile; gradient: 10-60%B/15 min., 60-10% B/2 min., 10% B/3 min.; 1.4 mL/min.; inj. vol.=10 μL;UV detection at 270 nm.

Retention times: Dimethylphosphonate 11=8.5 min., monomethyl phosphonate12=5.8 min.

The reaction solution was cooled slightly and the condenser was replacedwith a distillation head with thermometer, condenser and collectionflask (250 mL). The reaction solution was heated to reflux and 250 mL ofdistillate was collected. The pot solution was diluted withdichloromethane (250 mL) and an additional 250 mL of distillate wascollected. The distillation head was removed and the reaction flask wasplaced under nitrogen. The solution was diluted with dichloromethane(100 mL) and cooled to ice bath temperature. HPLC analysis of thereaction solution indicated the product at 89 Area %.

HPLC conditions:

YMC-Pack R & D, R-33-5 S-5 120A, 250×4.6 mm; mobile phase: Solvent A=20mM potassium phosphate, pH 6.2; Solvent B=acetonitrile; gradient: 10-60%B/15 min., 60-10% B/2 min., 10% B/3 min.; 1.4 mL/min.; inj. Vol=10 μL;UV detection at 270 nm.

Retention times: Product 8=8.5 min., starting material 7=5.9 min

Pyridine (18 mL) was added slowly to the stirred solution. After theaddition was complete (5 minutes), the resulting pale orange solutionwas stored at ice bath temperature until used (30 minutes).

Example 8.2 Coupling Reaction

A 2 L, 3-neck round bottom flask was equipped with a mechanical stirrer,and addition funnel (1 L). The flask was flushed with nitrogen andcharged with (S)-(−)-(3-chlorophenyl)-1,3-propanediol 3 (34.1 g), as asolution in dichloromethane (500 mL) and triethylamine (125 ml). Athermocouple probe was immersed in the reaction solution and the stirredcontents were cooled to −71° C. (dry ice/isopropanol). The dichloridatesolution 8 was charged to the addition funnel, then added slowly withstirring, maintaining the temperature <−67° C. After the addition wascomplete (1.25 h), the cooling bath was removed and the stirred mixturewas warmed to 0° C. over 30 min. The reaction mixture was washed withwater (550 mL) and the layers were separated. The dichloromethane phasewas diluted with ethyl acetate (500 mL) and washed with 5% NaCl solution(600 mL). The organic phase was dried (MgSO₄, 50 g), filtered throughdiatomaceous earth (Celite 521), and concentrated under reduced pressureto provide 108 g of a dark red sludge. The sample was dissolved inmethanol.

HPLC conditions:

YMC-Pack R & D, R-33-5 S-5 120A, 250×4.6 mm; mobile phase: Solvent A=20mM potassium phosphate, pH 6.2; Solvent B=acetonitrile; gradient: 10-60%B/15 min., 60-10% B/2 min., 10% B/3 min.; 1.4 mL/min.; inj. vol.=10 μL;UV detection at 270 nm.

Retention times: cis 13=12.5 min., trans 14=13.0 min.

The material was dissolved in ethanol (500 mL) and transferred to a 2 Lround bottom flask equipped with magnetic stirring, condenser andheating mantle. Acetic acid (55 mL) was added and the red solution washeated at reflux for 8 hours. HPLC indicated the reaction was complete.The sample was dissolved in methanol.

HPLC conditions:

YMC-Pack R & D, R-33-5 S-5 120A, 250×4.6 mm; mobile phase: Solvent A=20mM potassium phosphate, pH 6.2; Solvent B=acetonitrile; gradient: 10-60%B/15 min., 60-10% B/2 min., 10% B/3 min.; 1.4 mL/min.; inj. vol.=10 μL;UV detection at 270 nm. 6.

Retention times: cis 15=9.5 min., trans 16=9.8 min.

Example 8.3 Crystallization of9-{2-[2,4-cis(S)-(+)-4-(3-Chlorophenyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]methoxyethyl}adeninemethanesulfonate (9)

Methanesulfonic acid (21.5 mL) was added and a precipitate formed after15 min. The mixture was diluted with ethanol (400 mL) and heated untilall solids dissolved (pot temperature=70° C.). The solution was cooledwith stirring and a precipitate formed at 46° C. The resulting mixturewas stirred for 2 h, with cooling to ambient temperature, then at icebath temperature for 2.5 h. The mixture was filtered and the collectedsolid was washed with ethanol (2×15 mL) and dried to constant weight(−30 in. Hg, 55° C., 14 h). Recovery=49.4 g of a white powder 9 (51.9%).The solid contained 6.5 Area % of the trans diastereomer.

Chiral HPLC: Pirkle covalent (S,S) Whelk-O 1 10/100 krom FEC 250×4.6 mm;mobile phase=55:45, methanol: 0.1% HOAc in water; isocratic; 1.0mL/min.; inj. Vol.=10 μL; UV detection at 260 nm; sample preparation=2.0mg/mL in water. Retention times: cis-(R) 5=24.6 min., trans-(R) 6=27.5min., cis-(S) 7=18.0 min.

¹H-NMR (D₂O) was used to confirm structure of components.

Example 8.4 Recrystallization of9-{2-[2,4-cis-(S)-(+)-4-(3-Chlorophenyl)-2-oxo-1,3,2-dioxaphosphorinan-2-yl]methoxyethyl}adeninemethanesulfonate (9)

A 3 L, 3-neck round bottom flask was equipped with a mechanical stirrer,condenser, heating mantle and thermometer. The flask was charged with 2batches of crude mesylate salt 9 and ethanol (1.4 L). The stirredmixture was heated at reflux (pot temperature was 78° C.) until allsolids dissolved (approximately 10 minutes). The stirred mixture wasgradually cooled to ambient temperature over 1.5 hours (a precipitateformed at 56° C). The mixture was stirred at ambient temperature for anadditional 2 hrs., then filtered. The collected solid was washed withethanol (2×15 mL) and dried to constant weight (−30 in Hg, 65° C., 60hrs.).

Color: off white granular solid

Purity=97% (HPLC)

Optical purity (Chiral HPLC) >99.5%.

M.P.(° C.): 186.5-188

Specific Rotation (MeOH, 25° C., 589 nm): +16.429

Composition: C, 41.58; H, 4.56; N, 13.37 [Theoretical: C, 41.50; H,4.53; N, 13.35]

¹H NMR (D₂O): δ=1.30-1.60 (m, 1H), 1.80-1.95 (m, 1H), 2.60 (s, 3H),3.70-3.90 (m, 4H),4.10-4.50 (m, 2H), 4.60 (s, 3H), 5.15-5.40 (m, 1H),6.70-6.80 (m, 2H), 7.00-7.10 (m, 2H), 8.00 (s, 1H), 8.10 (s, 1H).

Example 9 Formation of Hydrochloride Salt via Salt Exchange

In this instant invention the oxalate salt of the phosphonic acid basedprodrugs was also formed. This salt form of the prodrug could beexchanged for other salts that are pharmaceutically acceptable. Theoxalate salt is dissolved in a solution containing an acid with a higherpK_(a), the acid dissociation constant.

A 3-neck round bottom flask is equipped with a mechanical stirrer,condenser, heating mantle and thermometer. The flask is charged withcrude oxalate salt and ethanol (5-10% solution by weight). The stirredmixture is heated at reflux (pot temperature is 78° C.) until all solidsdissolve. The solution is acidified with HCl and the stirred mixture isgradually cooled to ambient temperature (a precipitate forms as thetemperature cools). The mixture is stirred at ambient temperature withfiltration following. The collected solid consisting of thehydrochloride salt is washed with ethanol and is dried in a vacuum ovento constant weight (oven temperature=65° C.).

Example 10 Formation of the Sulfate Salt via Salt Exchange

A 3-neck round bottom flask is equipped with a mechanical stirrer,condenser, heating mantle and thermometer. The flask is charged withcrude mesylate salt 9 and ethanol (5-10% solution by weight). Thestirred mixture is heated at reflux (pot temperature is 78° C.) untilall solids dissolve. The solution is acidified with sulfuric acid andthe stirred mixture is gradually cooled to ambient temperature (aprecipitate forms as the temperature decreases). The mixture is stirredat ambient temperature and filtration of desired product follows. Thecollected solid consisting of the sulfate salt is washed with ethanoland is dried in a vacuum oven to constant weight (oven temperature=65°C.).

Example 11 Formation of the Sulfate Salt via Free Base Reaction

A 3-neck round bottom flask is equipped with a mechanical stirrer,condenser, heating mantle and thermometer. The flask is charged withcrude mesylate salt 9 and NaHCO₃ solution. The stirred mixture is heateduntil all solids dissolve. The solution is acidified with sulfuric acidand the stirred mixture is gradually cooled to ambient temperature (aprecipitate forms as the temperature decreases). The mixture is stirredat ambient temperature followed by filtration. The collected solidconsisting of the sulfate salt is washed with ethanol and is dried in avacuum oven to constant weight (oven temperature=65° C.).

Example 12 Formation of the Maleate Salt via Anionic Resin Reaction

A 3-neck round bottom flask is equipped with a mechanical stirrer,condenser, heating mantle and thermometer. The flask is charged withcrude mesylate salt 9. The stirred mixture is heated until all solidsdissolve. The mixture containing the mesylate salt 9 is run through ananionic resin. The resultant solution containing the free base of thecompound of Formula 1 is acidified with maleic acid and the stirredmixture is gradually cooled to ambient temperature (a precipitate formsas the temperature decreases). The mixture is stirred at ambienttemperature followed by filtration. The collected solid consisting ofthe maleate salt is washed with ethanol and is dried in a vacuum oven toconstant weight (oven temperature=65° C.).

1. A method for the preparation of compounds of Formula I:

wherein: M and V are cis to one another and MPO₃H₂ is a phosphonic acidselected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is phenyl,optionally substituted with 1-2 substituents selected from a groupconsisting of fluoro, chloro, and bromo; comprising: (a) coupling achiral 1-arylpropane-1,3-diol, wherein the aryl is a phenyl optionallysubstituted with 1-2 substituents selected from the group consisting offluoro, chloro, and bromo, with MPOCl₂ or an N-6 substituted analoguethereof.
 2. The method of claim 1 wherein the cis isomer has adiastereomeric excess of at least 50%.
 3. The method of claim 2 furthercomprising adding acid to form an acid addition salt of compounds ofFormula I.
 4. The method of claim 3 wherein said acid is selected fromthe group consisting of HCl, HBr, acetic acid, citric acid, maleic acid,methanesulfonic acid, nitric acid, phosphoric acid, succinic acid,sulfuric acid, and tartaric acid.
 5. The method of claim 3 wherein saidacid is selected from a group consisting of methanesulfonic acid,succinic acid, citric acid, and oxalic acid.
 6. The method of claim 5wherein said acid is methanesulfonic acid.
 7. The method of claim 3further comprising crystallizing said acid addition salt.
 8. The methodof claim 7 wherein the solvent for crystallizing said acid addition saltis selected from a group consisting of methanol, ethanol, isopropanol,acetone, toluene, and mixtures thereof.
 9. The method of claim 3 furthercomprising: (a) reacting a first acid addition salt compound of FormulaI with a second acid that has a higher acid dissociation constant thanthe first acid, and (b) crystallizing the desired second acid additionsalt compound of Formula I.
 10. The method of claim 3 furthercomprising: (a) neutralizing a first acid addition salt compound ofFormula I, (b) obtaining the free base of the compound of Formula I, (c)adding a pharmaceutically acceptable acid, and (d) crystallizing thedesired second acid addition salt compound of Formula I.
 11. The methodof claim 3 further comprising: (a) utilizing an anionic resin to obtainfree base of a first acid addition salt compound of Formula I, (b)adding a pharmaceutically acceptable acid, and (c) crystallizing thedesired second acid addition salt compound of Formula I.
 12. The methodof claim 1 wherein the reaction solution for the coupling step is at orbelow −50° C.
 13. The method in claim 12 wherein said reaction solutionis at or below −70° C.
 14. The method as recited in claim 1 wherein saidMPOCl₂ is added to said chiral 1-phenylpropane-1,3-diol.
 15. The methodof claim 1 wherein said MPOCl₂ is added to said chiral1-phenylpropane-1,3-diol at a reaction solution temperature at or below−50° C.
 16. The method of claim 1 further comprising addition of a baseto the reaction solution.
 17. The method of claim 1 wherein the MPOCl₂has an N-6 substituent to form a dialkylaminomethyleneimine.
 18. Themethod of claim 17 wherein said N-6 substituent is produced as part ofthe reaction to form the dichloridate.
 19. The method of claim 17wherein said dialkylaminomethyleneimine is selected from the groupconsisting of dimethylaminomethyleneimine, diethylaminomethylene,dipropylaminomethyleneimine, dibutylaminomethyleneimine,N-piperidinomethyleneimine, and N-morpholinomethyleneimine.
 20. Themethod of claim 1 wherein the compounds of Formula I have thestereochemistry of Formula II:


21. The method of claim 20 wherein the temperature for the coupling stepis at or below −50° C.
 22. A method for the preparation of compounds ofFormula I:

wherein: M and V are cis to one another and MPO₃H₂ is a phosphonic acidselected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is 3-chlorophenyl;comprising: (a) coupling a chiral 1-(3-chlorophenyl)propane-1,3-diolwith MPOCl₂ or an N-6 substituted analogue thereof.
 23. The method ofclaim 22 wherein the cis isomer has a diastereomeric excess of at least50%.
 24. The method of claim 23 further comprising adding acid to forman acid addition salt of Formula I.
 25. The method of claim 24 whereinsaid acid is selected from the group consisting of methanesulfonic acid,succinic acid, citric acid, and oxalic acid.
 26. The method of claim 25wherein said acid is methanesulfonic acid.
 27. The method of claim 22wherein the reaction solution for the coupling step is at or below −50°C.
 28. The method of claim 27 wherein said reaction solution is at orbelow −70° C.
 29. The method of claim 22 wherein said MPOCl₂ is added tosaid chiral 1-(3-chlorophenyl)propane-1,3-diol.
 30. The method of claim22 wherein the compounds of Formula I have the stereochemistry ofFormula II:


31. The method of claim 30 wherein the temperature for the coupling stepis at or below −50° C.
 32. The method of claim 30 further comprisingadding acid to form an acid addition salt of Formula II.
 33. The methodof claim 32 wherein said acid is selected from the group consisting ofmethanesulfonic acid, succinic acid, citric acid, and oxalic acid. 34.The method of claim 33 wherein said acid is methanesulfonic acid. 35.The method of claim 24 further comprising crystallizing said acidaddition salt from a solvent selected from the group consisting ofmethanol, ethanol, isopropanol, toluene, acetone, and mixtures thereof.36. The method as recited in claim 22 wherein the MPOCl₂ has an N-6substituent to form a dialkylaminomethyleneimine.
 37. The method asrecited in claim 36 wherein the N-6 substituent is produced as part ofthe reaction to form the dichloridate.
 38. The method as recited inclaim 36 wherein the dialkylaminomethyleneimine is selected from a groupconsisting of dimethylaminomethyleneimine, diethylaminomethylene,dipropylaminomethyleneimine, dibutylaminomethyleneimine,N-piperidinomethyleneimine, and N-morpholinomethyleneimine.
 39. A methodfor the preparation of compounds of Formula I:

wherein: M and V are cis to one another and MPO₃H₂ is phosphonic acidselected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine, and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is 2-bromophenyl;comprising: (a) coupling a chiral 1-(2-bromophenyl)propane-1,3-diol withMPOCl₂ or an N-6 substituted analogue thereof.
 40. The method of claim39 wherein the cis isomer has a diastereomeric excess of at least 50%.41. The method of claim 40 further comprising adding acid to form anacid addition salt of Formula I.
 42. The method of claim 41 wherein saidacid is selected from the group consisting of methanesulfonic acid,succinic acid, citric acid, and oxalic acid.
 43. The method of claim 42wherein said acid is methanesulfonic acid.
 44. The method of claim 39wherein the reaction solution for the coupling step is at or below −50°C.
 45. The method of claim 44 wherein said reaction solution is at orbelow −70° C.
 46. The method of claim 39 wherein said MPOCl₂ is added tosaid chiral 1-(2-bromophenyl)propane-1,3-diol.
 47. The method of claim39 wherein the compounds of Formula I have the stereochemistry ofFormula II:


48. The method of claim 40 wherein the temperature for the coupling stepis at or below −50° C.
 49. The method of claim 47 further comprisingadding acid to form an acid addition salt of Formula II.
 50. The methodof claim 49 wherein said acid is selected from the group consisting ofmethanesulfonic acid, succinic acid, citric acid, and oxalic acid. 51.The method of claim 50 wherein said acid is methanesulfonic acid. 52.The method of claim 41 further comprising crystallization of said acidaddition salt from a solvent selected from the group consisting ofmethanol, ethanol, isopropanol, acetone, toluene, and mixtures thereof.53. The method of claim 39 wherein the MPOCl₂ has an N-6 substituent toform a dialkylaminomethyleneimine.
 54. The method of claim 53 whereinthe N-6 substituent is produced as part of the reaction to form thedichloridate.
 55. The method of claim 53 wherein thedialkylaminomethyleneimine is selected from the group consisting ofdimethylaminomethyleneimine, diethylaminomethylene,dipropylaminomethyleneimine, dibutylaminomethyleneimine,N-piperidinomethyleneimine, and N-morpholinomethyleneimine.
 56. A methodfor the conversion of acid addition salt compounds of Formula I:

wherein: M and V are cis to one another and MPO₃H₂ is a phosphonic acidselected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine, and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is phenyl,optionally substituted with 1-2 substituents selected from a groupconsisting of fluoro, chloro, and bromo; comprising: (a) reacting saidfirst acid addition salt compound of Formula I with a second acid thathas a higher acid dissociation constant than the first acid, and (b)crystallizing desired second acid salt compound.
 57. The method of claim56 wherein said second acid is selected from the group consisting ofHCl, HBr, acetic acid, citric acid, maleic acid, methanesulfonic acid,nitric acid, phosphoric acid, succinic acid, sulfuric acid, and tartaricacid.
 58. The method of claim 57 wherein said second acid is selectedfrom the group consisting of methanesulfonic acid, succinic acid, citricacid, and oxalic acid.
 59. The method of claim 58 wherein said secondacid is methanesulfonic acid.
 60. A method for the conversion of firstacid addition salt compounds of Formula I:

wherein: M and V are cis to one another and MPO₃H₂ is a phosphonic acidselected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine, and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is phenyl,optionally substituted with 1-2 substituents selected from a groupconsisting of fluoro, chloro, and bromo; comprising: (a) neutralizingsaid first acid addition salt compound of Formula I, (b) obtaining thefree base of the compound of Formula I, (c) adding pharmaceuticallyacceptable acid, and (d) crystallizing desired second acid saltcompound.
 61. The method of claim 60 wherein the acid is selected fromthe group consisting of said pharmaceutically acceptable HCl, HBr,acetic acid, citric acid, maleic acid, methane sulfonic acid, nitricacid, phosphoric acid, succinic acid, sulfuric acid, and tartaric acid.62. A method for the conversion of a first acid addition salt compoundsof Formula I:

wherein: M and V are cis to one another and MPO₃H₂ is a phosphonic acidselected from the group consisting of9-(2-phosphonylmethoxyethyl)adenine, and(R)-9-(2-phosphonylmethoxypropyl)adenine; wherein V is phenyl,optionally substituted with 1-2 substituents selected from a groupconsisting of fluoro, chloro, and bromo; comprising: (a) utilizing ananionic resin to obtain the free base of the compound of Formula I, (b)adding a pharmaceutically acceptable acid, and (c) crystallizing thedesired second acid addition salt compound of Formula I.
 63. The methodof claim 62 wherein said pharmaceutically acceptable acid is selectedfrom the consisting of HCl, HBr, acetic acid, citric acid, maleic acid,methanesulfonic acid, nitric acid, phosphoric acid, succinic acid,sulfuric acid, and tartaric acid.